I'm trying to design a fluorescence filter set to view GFP fusion proteins
in live cells. I want to illuminate a narrow wavelength range centered on
the 488 488nm excitation peak of S65T. I'm considering an excitation
bandpass of 490 +/- 10nm, 505nm dichroic and 535 +/- 25nm emission
filters. My intention is to reduce phototoxicity of live cells by reducing
illumination at shorter wavelengths. But I could get in trouble with such
narrow excitation range if excitation or emission spectra are changed upon
fusion of GFP to other proteins, or when the fusion is in particular
intracellular environments. Does anyone know whether GFP spectra change
much? Other suggestions? Thanks!
--David Roof
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David Roof, Ph.D. Phone: 215-573-3636
Department of Physiology FAX: 215-573-5851
D202 Richards email: Roof at mail.med.upenn.edu
University of Pennsylvania
3700 Hamilton Walk
Philadelphia, PA 19104-6085
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